Device for depletion of the leucocyte content of blood and blood components

ABSTRACT

A high efficiency leucocyte-depletion filter for use with packed red cell concentrate derived from freshly drawn blood comprises a fibrous filter medium with a pore size of from about 0.5 to less than 3.6 μm and a CWST of from 53 to about 80. The filter is preferably used in conjunction with a gel prefilter and, optionally, a microaggregate filter so as to minimize clogging. In a preferred embodiment, the voids volume is about 60% to about 85%.

This application is a continuation of application Ser. No. 08/071,990,filed Jun. 7, 1993, now U.S. Pat. No. 5,344,561, which is a continuationof Ser. No. 07/719,506, filed Jun. 24, 1991, now U.S. Pat. No.5,229,012, which is a continuation-in-part of U.S. patent applicationSer. No. 07/527,717, filed May 22, 1990, now abandoned, which, in turn,is a continuation-in-part of U.S. patent application Ser. No.07/349,439, filed May 9, 1989 (now abandoned).

TECHNICAL FIELD

This invention relates to a method for depleting the leucocyte contentof whole blood and products derived therefrom, particularly from humanpacked red blood cells, and more particularly from anti-coagulated humanpacked red blood (PRC) cells which have been derived from whole bloodfreshly drawn from a blood donor.

BACKGROUND OF THE INVENTION

It has been the practice for 50 years or more to transfuse whole blood,and more recently blood components, from one or more donors to otherpersons. With the passage of time and accumulation of research andclinical data, transfusion practices have improved greatly. One aspectof current practice is that whole blood is rarely administered; rather,patients needing red blood cells are given packed red cells (hereinafterPRC), and patients needing platelets are given platelet concentrate.These components are separated from whole blood by centrifuging, theprocess providing, as a third product, plasma, from which various otheruseful components are obtained. In addition to the three above-listedcomponents, whole blood contains white blood cells (known collectivelyas leucocytes) of various types, of which the most important aregranulocytes and lymphocytes. White blood cells provide protectionagainst bacterial and viral infection.

In the mid to late seventies, a number of investigators proposed thatgranulocytes be separated from donated blood and transfused intopatients who lacked them, for example, those whose own cells had beenoverwhelmed by an infection. In the resulting investigations, it becameapparent that this practice is generally harmful, since patientsreceiving such transfusion developed high fevers, had other adversereactions, and often rejected the transfused cells. Further, thetransfusion of packed cells or whole blood containing donor leucocytescan be harmful to the recipient in other ways. Some of the viraldiseases induced by transfusion therapy, e.g., Cytomegaloviral InclusionDisease, which is a life threatening infection to newborns anddebilitated adults, are transmitted by the infusion of homologousleucocytes. Another life-threatening phenomenon affectingimmunocompromised patients is Graft versus host disease (GVH); a diseasein which the transfused leucocytes actually cause irreversible damage tothe blood recipient's organs including the skin, gastrointestinal tractand neurological system. More recently, retroviruses such as HIV (AIDS)and HTLV1 have become a threat. Since some viruses, including several ofthose described above, are resident in the leucocytes, the removal ofleucocytes is regarded as beneficial.

Conventional red cell transfusions have also been indicted as adverselyinfluencing the survival of patients undergoing surgery for malignancyof the large intestine. It is believed that this adverse effect ismediated by the transfusion of agents other than donor red blood cells,including the donor's leucocytes.

Removal of leucocytes to sufficiently low levels to prevent theundesired reactions, particularly in packed red cells which have beenderived from freshly drawn blood, is an objective of this invention.

In the currently used centrifugal methods for separating blood into thethree basic fractions (packed red cells, platelet concentrate, andplasma), the leucocytes are present in substantial quantities in boththe packed red cells and platelet concentrate fractions. It is nowgenerally accepted that it would be highly desirable to reduce theleucocyte concentration of these blood components to as low a level aspossible. While there is no firm criterion, it is generally acceptedthat many of the undesirable effects of transfusion would be reduced ifthe leucocyte content were reduced by a factor of about 100 or moreprior to administration to the patient. This approximates reducing thetotal content of leucocytes in a single unit of PRC (the quantity of PRCobtained from a single blood donation) to less than about 1×10⁷.Recently it has become more widely perceived that in order to preventviral infection by transfused blood, factors of reduction should be morethan 100, preferably more than 1000, and more preferably 30,000 or100,000 fold or more, such as 1,000,000 fold.

One of the most effective means of reducing leucocyte content that hasbeen discovered hitherto is disclosed in U.S. Pat. No. 4,925,572(Application Ser. No. 07/259,773, filed Oct. 19, 1988), which isdirected towards the bedside filtration of PRC. By contrast, thisinvention relates to the filtration of freshly drawn whole blood and offresh PRC, that is, PRC that is filtered within 24 hours, and morepreferably within 6 hours, of the time the blood was drawn. The behaviorof fresh PRC is very different from that of the 2 to 35 day old bloodthat is described in U.S. Pat. No. 4,925,572. The standards forleucocyte depletion are also very different; the above copendingapplication has as its objective leucocyte reduction by a factor of upto about 3000 to 10,000 and, while this is excellent for many purposes,the objective of the present application is leucocyte reduction by afactor in excess of about 30,000, and preferably of about 1,000,000 ormore.

Defining a Unit of Blood and a Unit of Packed Red Cells

Blood banks in the United States commonly draw about 450 milliliters(ml) of blood from the donor into a bag which usually contains ananticoagulant to prevent the blood from clotting. Herein the quantitydrawn during such a donation is defined as a unit of whole blood.

While whole blood is to a degree used as such, most units are processedindividually by centrifugation to produce one unit of PRC. The volume ofa unit of PRC varies considerably dependent on the hematocrit (percentby volume of red cells) of the drawn blood, which is usually in therange of about 37% to about 54%; and the hematocrit of the PRC, whichvaries over the range from about 50 to over about 80%, depending onwhether yield of one or another blood compound is to be minimized. MostPRC units are in the range of about 170 to about 350 ml, but variationbelow and above these figures is not uncommon.

Drawn whole blood may alternatively be processed by separating the redcells from the plasma, and resuspending them in a physiologicalsolution. A number of physiological solutions are in use. The red cellsso processed may be stored for a longer period before use, and with somepatients there may be some advantages in the removal of plasma. "Adsol"is the trade name of one such procedure, and SAG-M is a variant used inparts of Europe.

As used herein the term "fresh blood product" includes anti-coagulatedwhole blood, packed red cells obtained therefrom, and red cellsseparated from plasma and resuspended in physiological fluid, in allcases processed including filtration within about 24 hours andpreferably within 6 hours of when the blood was drawn.

In parts of the world other than the United States, blood banks andhospitals may draw less or more than about 450 ml of blood; herein,however, a "unit" is always defined by the United States' practice, anda unit of PRC or of red cells in physiological fluid is the quantityderived from one unit of whole blood.

As used herein, PRC refers to the blood products described above, and tosimilar blood products obtained by other means and with similarproperties.

Previously Available Means to Remove Leucocytes from PRC

The Spin-Filter system for obtaining leucocyte depleted packed red cellsis described by Parravicini, Rebulla, Apuzzo, Wenz and Sirchia inTransfusion 1984; 24:508-510, and is compared with other methods by Wenzin CRC Critical Reviews in Clinical Laboratory Sciences 1986; 24:1-20.This method is convenient and relatively inexpensive to perform; it hasbeen and continues to be used extensively. However, the efficiency ofleucocyte removal, while generally about 90% or better, is notsufficiently high to prevent adverse reactions in some patients.

Centrifugation methods are available which produce lower levels ofleucocytes in red cells, but these are laboratory procedures which arevery costly to operate, and sterility of the product is compromised to adegree such that it must be used within 24 hours.

Other methods for leucocyte depletion, such as saline washing ordeglycerolizing frozen red cells, have been or are used, but these havedisadvantages for economical, high reliability service.

A number of devices have been proposed in which fibers are packed intohousings, and whole blood passed through them in order to removemicroaggregates and a portion of the leucocyte content. These deviceshave, when reduced to practice, all required saline to be applied eitherbefore or after use, or both before and after use, and are very poorlysuited for blood bank use.

Characteristics Desirable in a Leucocyte Depletion Device

An ideal device for leucocyte depletion intended for use by blood bankswould be inexpensive, relatively small, and be capable of processing oneunit of PRC rapidly, for example in less than about one hour, and reducethe leucocyte content to the lowest possible level. Because of the highcost and limited availability of red blood cells, this ideal devicewould deliver the highest possible proportion of the red cells presentin the donated blood. Such a device is an object of this invention.

Devices which have previously been developed in attempts to meet thisobjective have been based on the use of packed fibers, and havegenerally been referred to as filters. However, it would appear onpreliminary review that processes utilizing filtration based onseparation by size cannot succeed for two reasons. First, the varioustypes of leucocytes range from granulocytes and macrocytes, which can belarger than about 15 μm, to lymphocytes, which are in the 5 to 7 μmrange. Together, granulocytes and lymphocytes represent the majorproportion of all of the leucocytes in normal blood. Red blood cells areabout 7 μm in diameter, i.e., in size they are in the range of one ofthe two major components which must be removed. Secondly, all of thesecells deform so as to pass through much smaller openings than theirnormal size. Accordingly, and because it is readily observed bymicroscopic examination that leucocytes are absorbed on a variety ofsurfaces, it has been widely accepted that removal of leucocytes isaccomplished mainly by adsorption, rather than by filtration. Anunexpected and surprising result of this invention, however, is thatfiltration through certain filters having a controlled pore size iscritical to reach the target levels of leucocyte depletion.

Blood Component Recovery

In the preceding section, reference was made to the desirability ofrecovering a high proportion of the red cells delivered to theseparation device. There are several causes for reduced recovery of redcells:

(a) Losses due to hold up within the connecting tubing;

(b) Losses due to liquid which remains within the device itself at theconclusion of the filtration;

(c) Losses due to adsorption on the surfaces of the device, or due tomechanical entrapment within the device;

(d) Loss due to clogging of the filter prior to completion of thepassage of the full unit of blood; and

(e) Losses due to contact with incompatible surfaces, which can causeclotting.

Capacity

As separated from whole blood in current blood banking practice, packedred cells contain not only a proportion of the leucocytes present in theblood as drawn from the donor, but also some platelets (which tend to bevery adhesive), fibrinogen, fibrin strands, tiny fat globules, andnumerous other components normally present in small proportions. Alsocontained are factors added at the time the blood is drawn to preventclotting, and nutrients which help to preserve the red cells duringstorage.

During the centrifuging process which concentrates the red cells andpartially separates them from the remaining components there is atendency for microaggregates to form in PRC. These may comprise some redcells together with leucocytes, platelets, fibrinogen, fibrin, fat, andother components. Gels, which may be formed by fibrinogen and/or fibrin,may also be present in PRC produced by blood banks.

If the leucocyte depletion device comprises a porous structure,microaggregates, gels and occasionally fat globules tend to collect onor within the pores, causing blockage which inhibits flow.

Ease and Rapidity of Priming

Ease of use is an important characteristic of any leucocyte depletionsystem. As noted above, for leucocyte depletion devices, ease of primingis a particularly important factor. The term "priming time" refers tostart-up of flow of PRC from the bag through the filter to the patient,and is the time required to fill the filter housing from its inlet toits outlet. An object of this invention is to maintain a short primingtime, preferably less than about 30 to about 120 seconds, to conservetechnician time.

Preconditioning of Leucocyte Depletion Devices Prior to Priming

A number of devices in current use require pretreatment prior to passingblood, usually consisting of passing physiological saline. The necessityfor such an operation is very undesirable in blood bank processingbecause it complicates the procedure, requires technician time, and putsmaintenance of sterility at risk.

The reasons for using such pretreatment vary. They include removal ofacid hydrolysate developed during steam sterilization of devicescontaining cellulose acetate fibers, assurance of freedom from foreignsolids which may be present in natural fibers, and if the fibers arehygroscopic to prevent hemolysis (loss of the integrity of red bloodcells with subsequent loss of their contents to the external milieu).

An objective of this invention is a leucocyte depletion device whichrequires no preconditioning prior to processing PRC derived from freshlydrawn blood.

Definition of Voids Volumes

The concept of "voids volume" is related to, but distinguishable from,the term "bulk density". In fact, the term bulk density is misleadingwhen referring to a broad spectrum of fibers with large variations inspecific gravity. For example, polyester fibers may have a specificgravity of about 1.38 while inorganic fibers prepared from zirconia mayhave a specific gravity of greater than 5. Thus, in carrying out theinstant invention, references to voids volume should not be confusedwith the term bulk density.

The concept of voids volume may be explained as follows.

Calculation of Voids Volume, Given Bulk Density and Fiber Density

Bulk density, D, is the weight of a given volume of fibrous aggregatedivided by its apparent volume. Normally this is expressed in g/cc.

By fibrous aggregate is meant one or more fibers occupying a given orapparent volume, e.g., a mass of non-woven intertangled fibers with acertain proportion of voids or spaces within the mass.

In order to calculate the voids volume, V, the density, d, of the fibersmust be known. The density, d, is also expressed in g/cc.

1. The volume of 1 gram of fibrous aggregate=1/D

2. The volume of 1 gram of fibers=1/d

3. The voids volume, V, is the total aggregate volume less the fibervolume or ##EQU1##

EXAMPLE

Given:

Volume of fibrous aggregate=10 cc

Weight of aggregate=1 g

Density of the aggregate D=1/10=0.1 g/cc

Density of fiber d=1.38 ##EQU2##

The following table illustrates the difference between specifying voidsvolume and density. As illustrated there, at constant density, a columnof glass fibers (glass being much more dense than, e.g., polypropylene)has a voids volume of 94% versus only 83.3% for a column ofpolypropylene.

    ______________________________________    D, Column  Material     Density of                                      Voids    density    of the       the fiber,                                      Volume    (g/cc)     fiber        (g/cc)    (%)    ______________________________________    0.15       glass*       2.5       94.0    0.15       polyester    1.38      89.1    0.15       polypropylene                            0.9       83.3    ______________________________________     *Glass varies in density from about 2.3 to about 2.7 g/cc. The 2.5 g/cc     figure used here is in the mid range.

Definition of Pore Diameter

In the definition of various filter media, it will be necessary to usethe term "pore diameter". This term as used herein is as determined bythe modified OSU F2 test described below.

Wetting of Fibrous Media

When a liquid is brought into contact with the upstream surface of aporous medium and a small pressure differential is applied, flow intoand through the porous medium may or may not occur. A condition in whichno flow occurs is that in which the liquid does not wet the material ofwhich the porous structure is made.

A series of liquids can be prepared, each with a surface tension ofabout 3 dynes/cm higher compared with the one preceding. A drop of eachmay then be placed on a porous surface and observed to determine whetherit is absorbed quickly, or remains on the surface. For example, applyingthis technique to a 0.2 μm porous tetrafluoroethylene (PTFE) filtersheet, instant wetting is observed for a liquid with a surface tensionof about 26 dynes/cm. However, the structure remains unwetted when aliquid with a surface tension of about 29 dynes/cm is applied.

Similar behavior is observed for porous media made using other syntheticresins, with the wet-unwet values dependent principally on the surfacecharacteristics of the material from which the porous medium is made,and secondarily, on the pore size characteristics of the porous medium.For example, fibrous polyester, specifically polybutylene terephthalate(hereinafter "PBT") sheets which have pore diameters less than about 20μm will be wetted by a liquid with a surface tension of about 50dynes/cm, but will not be wetted by a liquid with a surface tension ofabout 54 dynes/cm.

In order to characterize this behavior of a porous medium, the term"critical wetting surface tension" (CWST) is defined as follows. TheCWST of a porous medium may be determined by individually applying toits surface a series of liquids with surface tensions varying by about 2to about 4 dynes/cm, and observing the absorption or non-absorption ofeach liquid. The CWST of a porous medium, in units of dynes/cm, isdefined as the mean value of the surface tension of the liquid which isabsorbed and that of a liquid of neighboring surface tension which isnot absorbed. Thus, in the examples of the two preceding paragraphs, theCWST's are, respectively, about 27.5 and about 52 dynes/cm.

In measuring CWST, a series of standard liquids for testing is preparedwith surface tensions varying in a sequential manner by about 2 to about4 dynes/cm. Ten drops of each of at least two of the sequential surfacetension standard liquids are independently placed on representativeportions of the porous medium and allowed to stand for 10 minutes.Observation is made after 10 minutes. Wetting is defined as absorptioninto or obvious wetting of the porous medium by at least nine of the tendrops within 10 minutes. Non-wetting is defined by non-absorption ornon-wetting of at least nine of the ten drops in 10 minutes. Testing iscontinued using liquids of successively higher or lower surface tension,until a pair has been identified, one wetting and one non-wetting, whichare the most closely spaced in surface tension. The CWST is then withinthat range and, for convenience, the average of the two surface tensionsis used as a single number to specify the CWST.

A number of alternative methods for contacting porous media with liquidsof sequentially varying surface tension can be expected to suggestthemselves to a person knowledgeable of physical chemistry after readingthe description above. One such involves floating a specimen on thesurfaces of liquids of sequentially varying surface tension values, andobserving for wet-through of the liquid, or if the fiber used is moredense than water, observing for sinking or floating. Another means wouldclamp the test specimen in a suitable jig, followed by wetting with thetest liquids while applying varying degrees of vacuum to the undersideof the specimen.

Appropriate solutions with varying surface tension can be prepared in avariety of ways, however, those used in the development of the productdescribed herein were:

    ______________________________________                         Surface Tension    Solution or fluid    range, dynes/cm    ______________________________________    Sodium hydroxide in water                         94-110    Calcium chloride in water                         90-94    Sodium nitrate in water                         75-87    Pure water           72.4    Acetic acid in water 38-69    Ethanol in water     22-35    n-Hexane             18.4    FC77 (3M Corp.)      15    FC84 (3M Corp.)      13    ______________________________________

Wetting of Fibrous Media by Blood

In packed red cells, as well as in whole blood, the red cells aresuspended in blood plasma, which has a surface tension of about 73dynes/cm. Hence, if packed red cells or whole blood is placed in contactwith a porous medium, spontaneous wetting will occur if the porousmedium has a CWST of about 73 dynes/cm or higher.

Hematocrit is the percent by volume occupied by red cells. Thehematocrit of packed red cells ranges from about 50 to above 80%. Thus,about 50 to over 80% of the volume of PRC consists of the red cellsthemselves and, for this reason, the surface characteristics of the redcells influence the wetting behavior of PRC. The surface tension hasbeen measured and is given in the literature as 64.5 dynes/cm.("Measurement of Surface Tensions of Blood Cells & Proteins", by A. W.Neumann et al., from Annals N.Y.A.S., 1983, pp. 276-297.) The lowersurface tension of red cells affects the behavior of PRC, for exampleduring priming of filters and during filtration, in ways which are notfully understood.

The benefits conferred by preconditioning fibers to CWST values higherthan the natural CWST of PBT and other synthetic fibers include:

(a) An important aspect of this invention is the discovery that fibrousmedia treated to convert the fiber surfaces to a particular range ofCWST perform better with respect to priming time, leucocyte depletionefficiency, and resistance to clogging than do fibrous media with CWSTvalues outside of those ranges.

(b) Synthetic fiber media whose CWST values have been elevated bygrafting have, when hot compressed, superior fiber-to-fiber bonding andare for this reason preferred for use in making the preformed elementsused in this invention.

(c) Detrimental effects such as occasional clotting of blood associatedwith non-wetting as described in previous sections are avoided.

(d) Devices made using unmodified synthetic fibers are recommended to beflushed with saline prior to use. This operation is undesirable since itcauses blood loss due to hold-up within the complex tubing arrangementrequired, adds to cost, operation time, and operation complexity, andincreases the probability that sterility may be lost. The need forpreflushing is obviated by raising the CWST to the values disclosed inthis invention.

Description Of The Invention

In accordance with the subject invention, a device and a method fordepleting the leucocyte content of a blood product are provided.

The invention comprises a device for the depletion of the leucocytecontent of fresh blood products which comprises a fibrous leucocyteadsorption/filtration filter with a pore diameter of from about 0.5 toless than 3.6 μm and having a CWST of from 53 to about 80 dynes/cm.

One of the significant advantages of the device of the invention relatesto the priming of the filter assembly (i.e., inducing sufficient flow ofPRC to fill the housing), which is more complex and more difficult thanwould appear at first sight.

If the CWST of the fiber surface is too low, for example that ofunmodified synthetic fiber, relatively higher pressure is required toforce the PRC to flow through. More seriously, areas of the filtermedium tend to remain unwetted, preventing flow of PRC. Further,clotting may occur, especially with finer, high surface area fibers andwith older blood.

For reasons which are not well understood, filters which have CWST inexcess of about 90 dynes/cm have been observed to have very long primingtimes, ranging to about 2 to about 5 minutes. It has further beenlearned, by trial and error, that it is advisable that the CWST be heldwithin a range somewhat above the CWST of untreated polyester fiber (52dynes/cm), for example, about 55 dynes/cm and higher, and below about 75or 80 dynes/cm, and more preferably from about 60 to about 70 dynes/cm.

The filter element of the invention has a pore size of from about 0.5 toless than 3.6 μm, preferably from about 0.5 to about 3.5 μm, morepreferably from about 0.5 to about 2 μm. This in itself is surprisingsince such pore sizes are significantly smaller than the size of bloodcomponents such as red blood cells which nevertheless pass through. Thepreferred element is typically made using 2.6 μm average fiber diameterradiation grafted melt blown polybutylene terephthalate (PBT) web, whichin a preferred form is hot compressed to a voids volume of about 60% toabout 85% and preferably about 65% to about 84% and has a pore diameterof about 0.5 to about 2 μm. The fiber surfaces of the adsorption elementare surface grafted to provide a CWST preferably in the range of about60 to about 70 dynes/cm, such as from about 62 to about 68 dynes/cm. Itmay be protected from clogging by a gel prefilter and/or by amicroaggregate prefilter, and its function is to reduce the leucocytecontent by a factor of 30,000 or more while allowing red cells to passfreely.

In a preferred device the fibrous filter medium of the invention ispreceded by one or two preformed elements. If a three element filter isused, the function of the first, (the gel prefilter), is to remove gels;that of the second, (the microaggregate filter), is primarily to removemicroaggregates though it can also remove some leucocytes by adsorptionand by filtration; and the function of the third, the filter medium ofthe invention (often called hereinafter the adsorption/filtrationfilter), is to remove leucocytes by adsorption and by filtration. Ifonly two elements are used, the first may be a gel prefilter or amicroaggregate filter, followed by the adsorption/filtration filter ofthe invention. Each of these three elements may comprise one or moreseparate or integral fibrous layers. The respective elements may differin their CWSTs, voids volumes, pore sizes, and number of layers. Eachelement may comprise one or more preforms each containing a number oflayers. The respective preforms within each element may also differ withrespect to the preceding characteristics.

Significant and novel preferred features of this invention whichcontribute to achieving high efficiency and capacity for leucocyteremoval, and minimize loss of blood within the apparatus include:

(a) Previously disclosed devices have used a relatively small crosssectional area perpendicular to the flow path, and are correspondinglylonger with respect to the depth of their flow paths. The preferreddevices in accordance with this invention are larger in cross sectionalarea perpendicular to the flow path and correspondingly shorter in depthof flow. This improvement in design helps to prevent clogging by PRCcontaining unusually high quantities of gels or microaggregates.

(b) In order to make the larger cross sectional area economic andpractical and to obtain the required degree of prefiltration, the filtercomponents used in accordance with this invention are preferablypreformed prior to assembly to closely controlled dimension and densityparameters so as to form, in whole or in part, integral elements,self-contained and independent of other elements until assembled into adevice in accordance with the subject invention. By "integral element"is meant a unitary, complete structure having its own integrity and, asmentioned, self-contained and independent of the other integral elementsuntil assembled.

Preforming eliminates the pressure on the inlet and outlet faces of thehousing which are inherent in a packed fiber system. Preforming alsopermits one element, for example, the first stage prefilter of theassembled device, to be more or less compressible, yet have a lower orhigher density than the one following it. This arrangement contributesto longer life in service.

Preforming makes it more practical to use larger cross sectional arealeucocyte depletion devices which have longer life in service, coupledwith at least equal and usually better leucocyte removal efficiency,equal or better red cell recovery, and less hold up, when compared withdevices that use fibers or fibrous webs packed into a housing atassembly.

Devices have been proposed and some made which comprise variouscommercially made woven and non-woven fibrous media as prefilters, alongwith a more finely pored last stage consisting of non-woven fibrousmats, all packed within a plastic housing. These devices have not hadthe efficient prefiltration made possible by preforming and, inaddition, have been prone to occasional clogging, being too small incross sectional area.

(c) While it might be thought that freshly drawn blood would be free ofaggregates and gels, hence prefiltration would not be required toprevent filter clogging, it has been the experience of the applicantsthat freshly drawn blood does occasionally clog a filter capable ofproducing a filtrate with less than about 10⁴ leucocytes per unit ofPRC, corresponding to a reduction in leucocyte content by a factor ofabout 10⁵.

Because of the difficulty of predicting the consequences of the unusualand variable combination of clogging factors that may be present, evenfor a person skilled in the art of filter design, it is advisable toincorporate an efficient prefilter.

The present invention, therefore, provides for the optional use of anefficient, small volume gel prefilter system which will contribute tothe objective of achieving an average reduction of leucocyte content bya factor of about 30,000 or more, while rarely or never clogging whenpassing one unit of packed red cells derived from freshly drawn blood.

The use of such an effective gel prefilter which consistently retains atleast a substantial proportion of the gel content of one unit of PRCderived from freshly drawn blood is, therefore, a preferred feature ofone aspect of this invention. This makes possible the use of a devicewith a smaller internal volume, with less blood loss due to internalhold-up, while consistently delivering one unit of PRC without clogging.

(d) While the gel prefilter is extremely efficient in removing gels witha very small increase in pressure drop, and frequently removes as wellquantities of microaggregates suspended in the gels, it removes only aportion of any microaggregates that may be present. Removal of thesmaller microaggregates may be accomplished by one, two, or more layersof prefiltration using filter media of intermediate pore diameter whichmay either be separate preformed layers, but which in a preferred formof this invention are integral with part or all of theadsorption/filtration element.

(e) The housing into which the element assembly is sealed is uniquelydesigned to achieve convenience of use, rapid priming, and efficient airclearance, this last leading to further reduction in hold-up of PRC.

(f) The lateral dimensions of the elements are larger than thecorresponding inner dimensions of the housing into which they areassembled. For example, if the elements are of disc form, the discoutside diameter is made about 0.1 to about 0.5% larger than the housinginside diameter. This provides very effective sealing by forming aninterference fit with no loss of effective area of the elements, andcontributes further towards minimization of the blood hold-up volume ofthe assembly.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross sectional view of an exemplary depletion deviceemploying the filter element of the present invention.

FIG. 2 is an elevation view of the inside surface of the inlet sectionof the depletion device shown in FIG. 1.

FIG. 3 is an elevation view of the inside surface of the outlet sectionof the depletion device shown in FIG. 1.

FIG. 4 is a cross sectional view of the outlet section shown in FIG. 3.

BEST MODE FOR CARRYING OUT THE INVENTION Material for Use inConstruction of Leucocyte Removal Devices

A variety of starting materials other than fibers can be considered; forexample, porous media could be cast from resin solution to make porousmembranes, or sintered metal powder or fiber media could be used.However, considerations of cost, convenience, flexibility, and ease offabrication and control, point to fibers as a preferred startingmaterial.

In order to achieve good priming with the fibrous medium fully wettedand in the absence of surfactant deliberately added to reduce thesurface tension of the blood product, it would appear at first glancefrom elementary consideration of the physical chemistry involved thatblood component devices should be made of materials which have CWSTvalues in the range of about 70 to about 75 dynes/cm or higher.Practical considerations dictate the use of commercially availablefibers. Synthetic resins from which fibers are prepared commerciallyinclude polyvinylidene fluoride, polyethylene, polypropylene, celluloseacetate, Nylon 6 and 66, polyester, polyacrylonitrile, and polyaramid.An important characteristic of resins is their critical surface tension(Zisman, "Contact angles, wettability and adhesion", Adv. Chem. Ser. 43,1-51, 1964). These resins have critical surface tensions (γ_(c)) rangingfrom about 25 to about 45 dynes/cm. Experience has shown that the CWSTof filters in the pore sizes preferred in the products of this inventioncan be expected to be less than about 10 dynes/cm higher than γ_(c). Forexample, for polytetrafluoroethylene, γ_(c) is 18 and CWST is 27.5,while for a polyester PBT fibrous mat, γ_(c) is 45, and CWST is 52. Nocommercially available synthetic fiber has been found which has a CWSThigher than about 52 dynes/cm.

Some natural fibers have CWST greater than 52, but natural fiberssmaller than about 15 μm in diameter are not generally commerciallyavailable. Synthetic fiber webs in which the fibers are less than about5 μm in diameter can be made by the melt blowing process, and comparedwith natural fibers, such fibers require one third or less the mass toprovide equal fiber surface area for adsorption of leucocytes, andconsequently, occupy less volume when fabricated into filters of a givenpore diameter. For this reason, natural fibers are less suited formanufacturing leucocyte removal devices with optimally low hold-upvolume. For example, a commercially available packed cotton fiber devicecurrently used for leucocyte depletion has a priming volume of over 75ml, which is more than twice the volume of the preferred devicedescribed in this application. Furthermore, the makers of this devicerequire saline to be passed before and after the PRC has been passed,and the device is not suitable for bedside use. Additionally, blood soprocessed must be used within 24 hours.

The art of surface grafting has been the subject of extensive researchfor 25 years or more. Numerous publications in the scientific literatureand a large number of patents describe a variety of methods andprocedures for accomplishing surface modification by this means. Onesuch method employs monomers comprising an acrylic moiety together witha second group which can be selected to vary from hydrophilic (e.g.,--COOH or --OH) to hydrophobic (e.g., saturated chains such as --CH₂ CH₂CH₃), and these have been used in the process of this invention. Heat,UV, and other reaction energizing methods can be used to initiate andcomplete the reaction. However, cobalt source radiation grafting hasbeen selected as most convenient and has been used in this invention tomodify the CWST of fibrous mats. By cut and try selection, mixtures ofmonomers or single monomers can be found which will produce a fibrousmat of polybutylene terephthalate in which the CWST has been increasedfrom 52 to any desired value up to as high as is possible to be measuredby the method described above. The upper limit is set by the paucity ofliquids with surface tensions at room temperature higher than about 110dynes/cm.

During the development of this invention, devices were prepared usingmedia in which grafting was accomplished by compounds containing anethylenically unsaturated group such as an acrylic moiety combined witha hydroxyl group (for example, 2-hydroxyethyl methacrylate, or "HEMA").A second acrylic monomer, such as methyl acrylate (MA) or methylmethacrylate (MMA), which tend to cause the grafted porous webs to havelower CWST, can be used in combination with HEMA, and by varying theproportions, any CWST between about 35 to about 45 to over 110 dynes percm can be obtained.

Liquids with surface tensions lower than the CWST of the porous mediumwill wet the medium and, if the medium has through pores, will flowthrough it readily. Liquids with surface tensions higher than the CWSTwill not flow at all at low differential pressures, but will do so ifthe pressure is raised sufficiently. If the surface tension of theliquid is only slightly above the CWST, the required pressure will besmall. If the surface tension differential is high, the pressurerequired to induce flow will be higher.

It has been discovered that, when a liquid is forced under pressure topass through a fibrous mat which has a CWST more than about 15 to about20 dynes/cm lower than the liquid's surface tension, flow tends to occurin a non-uniform fashion, such that some areas of the mat remain dry.This is highly undesirable in a leucocyte depletion device, firstbecause the pressure drop is higher causing earlier clogging, secondbecause all the flow passes through only a portion of the availablearea, again increasing the probability of clogging, and third becauseonly a portion of the fiber surface area available for adsorption of orretention by filtration of leucocytes is used for that purpose and, as aresult, leucocyte removal is less efficient.

Solutions to the Problems of Poor Wetting of Synthetic Fibers

Fiber surface characteristics of most or all of the synthetic resinslisted above, as well as of other materials, can be modified by a numberof methods, for example, by chemical reaction including wet or dryoxidation, by coating the surface by depositing a polymer thereon, andby grafting reactions which are activated by exposure to an energysource such as heat, a Van der Graff generator, ultraviolet light, or tovarious other forms of radiation, among which gamma-radiation isparticularly useful.

As examples of these various methods, stainless steel fibers can be madewater wettable, i.e., provided with a γ_(c) greater than about 72dynes/cm by oxidation in air at about 370° C. to produce a thin oxidesurface skin. Synthetic organic and glass fibers may be coated bypolymers which contain at one end a reactive (e.g., epoxide) moiety andat the other a hydrophilic group.

While the above methods and others known to those familiar with surfacemodification techniques can be used, radiation grafting, when carriedout under appropriate conditions, has the advantage that considerableflexibility is available in the kinds of surfaces that can be modified,in the wide range of reactants available for modification, and in thesystems available for activating the required reaction. In the subjectinvention gamma-radiation grafting has been focused on because of theability to prepare synthetic organic fibrous media with CWST over thefull range of from below about 50 to above 80 dynes/cm. The products arevery stable, have zero or near zero aqueous extractables levels and, inaddition, improved adhesion between fibers is obtained when used inpreformed prefiltration or in adsorption/filtration elements.

Selection of Fiber Diameter for Use in Leucocyte Depletion Devices

As noted in the section headed "Characteristics Desirable in a LeucocyteDepletion Device", adsorption of leucocytes on fiber surfaces is widelyaccepted as the mechanism of leucocyte removal. Since the surface areaof a given weight of fibers is inversely proportional to the diameter ofthe fibers, and removal of leucocytes by adsorption to the fibersurfaces is a significant mechanism for leucocyte depletion, it is to beexpected that finer fibers will have higher capacity and that thequantity, as measured by weight of fibers necessary to achieve a desiredefficiency, will be less if the fibers used are smaller in diameter.

For this reason and because it is well known that finer fibers quitegenerally contribute to higher efficiency and longer life of filters,the trend has been to use finer fibers for leucocyte depletion.Historically, as the technology required to produce smaller diameterfibers has advanced, they have soon thereafter been packed into housingsand/or proposed to be used for leucocyte depletion.

Selection of Fiber for Leucocyte Depletion Devices

A number of commonly used fibers, including polyesters, polyamides, andacrylics, lend themselves to radiation grafting because they haveadequate resistance to degradation by gamma-radiation at the levelsrequired for grafting, and they contain groups with which availablemonomers can react. Others, such as polypropylene, are less readilyadapted to modification by grafting.

As noted above, fiber diameters should be as small as possible.Synthetic fibers made by conventional spinneret extrusion and drawingare not currently available smaller than about 6 μm in diameter.

Melt blowing, in which molten resin is attenuated into fibers by a highvelocity stream of gas and collected as a non-woven web, came intoproduction in the 1960s and 1970s and has been gradually extended overthe years with respect to the lower limit of fiber diameter with whichwebs could be made. Within recent years, webs with fiber diameters lessthan 3 μm have been achieved, and more recently, webs of good qualitywith average fiber diameter less than 2 μm have been made.

Some resins are better adapted to melt blowing of fine fibers than areothers. Resins which work well include polypropylene, polymethylpentene,polyamides such as Nylon 6 and Nylon 66, polyester PET (polyethyleneterephthalate), and polyester PBT (polybutylene terephthalate). Othersmay exist that have not yet been tested. Of the above listed resins,polyester PBT is a preferred material because it lends itself toradiation grafting and to subsequent conversion into preformed elementsof controlled pore size by hot pressing.

Polyester PBT has been the principal resin used for the development ofthe products of this invention and is, except for a portion of a gelprefilter, the resin used in the examples. It should be noted, however,that other resins may be found which can be fiberized and collected asmats or webs with fibers as small as about 1.5 μm in diameter or less,and that such products, with their CWST adjusted if necessary to theoptimum range, may be well suited to the fabrication of equallyefficient but still smaller leucocyte depletion devices. Similarly,glass and other fibers, appropriately treated, may make suitable deviceswith very low hold-up of blood.

Description of an Exemplary Depletion Device

As shown in FIGS. 1-4, an exemplary depletion device 10 generallycomprises a housing 11 and a separation element or filter-adsorberassembly 12. The housing 11 has an inlet 13 and an outlet 14 and definesa fluid flowpath between the inlet 13 and the outlet 14. Thefilter-adsorber assembly 12 is disposed within the housing 11 across thefluid flowpath and serves to separate undesirable substances, such asgels, fat globules, aggregates, and leucocytes, from a fluid, such as asuspension of packed red cells, flowing through the housing 11.

Housings can be designed to accept a variety of shapes offilter-adsorber assemblies. One such is, for example, a square. Thoseand other possible forms would in principle all be functional, providedthat adequate flow area is provided.

A square filter-adsorber assembly would in theory allow more economicaluse of material, but would be less reliable if an interference fit sealwere used in the manner described below for housings fitted with discshaped filter-adsorber assemblies. If sealing is obtained by edgecompression about the periphery, significant effective area is lost atthe seal. For those reasons, cylindrical housings with disc shapedfilter-adsorber assemblies assembled with an interference fit seal arepreferred, although other forms may be used. Circular housings with aneffective cross sectional area of about 62 cm² have been used indeveloping this invention.

Housings can be fabricated from any suitably impervious material,including an impervious thermoplastic material. For example, the housingmay preferably be fabricated from a transparent polymer, such as anacrylic or polycarbonate resin, by injection molding. Not only is such ahousing easily and economically fabricated, but it also allowsobservation of the passage of the fluid through the housing. Thehousings are designed to withstand normal abuse during service, as wellas internal pressures up to about 3 psi (0.2 Kg/cm²). This permits lightconstruction, which is a desirable feature of this invention madepossible by the use of preformed filter-adsorber assemblies. The forcerequired to compress the fibers of an efficiently designedfilter-adsorber assembly by packing of fibers into a housing is as highas about 68 kilograms for a 62 cm² disc, or about 1.1 kg/cm², requiringheavier, bulkier, and more costly housing construction.

While the housing may be fashioned in a variety of configurations, thehousing 11 of the exemplary separation device 10 is preferably fashionedin two sections, i.e., an inlet section 15 and an outlet section 16. Theinlet section 15 includes a circular inlet plate 20, and the insidesurface of the circular inlet plate 20 defines a wall 21 which faces theupstream surface of the filter-adsorber assembly 12.

The inlet 13 delivers the fluid to an inlet plenum 22 between the wall21 and the upstream surface of the filter-adsorber assembly 12. Inaccordance with one aspect of the invention, the inlet 13 delivers thefluid to the inlet plenum 22 at or near the bottom of the housing 11, asshown in FIGS. 1 and 2.

The inlet may be variously configured. However, the inlet 13 of theexemplary separation device 10 includes a longitudinal inlet ridge 23.The inlet ridge 23 extends along the outside surface of the circularinlet plate 20 parallel to a diametrical axis A of the housing 11,which, in use, is positioned with the diametrical axis A orientedgenerally vertically. The upper end of the inlet ridge 23 may befashioned as a socket for receiving a hollow spike 24 which is used topierce the bottom of a bag containing the fluid, e.g., a blood bag. Theinlet 13 further includes an inlet passageway 25 which opens at theupper end of the hollow spike 24, extends through the hollow spike 24and the inlet ridge 23, and communicates with the inlet plenum 22 at thebottom of the inlet section 15.

The wall 21 of the circular inlet plate 20 includes a plurality ofgenerally concentric circular ridges 26 which define concentric circulargrooves 27. The ridges 26 abut the upstream surface of thefilter-adsorber assembly 12. As shown in FIG. 2, the ridges 26 terminatein the lower portion of the inlet section 15, defining a passageway oraccess 30. The access 30 extends between the inlet passageway 25 andeach circular groove 27, allowing fluid to flow from the inletpassageway 25 to the circular grooves 27. Collectively, the circulargrooves 27 and the access 30 define the inlet plenum 22, whichdistributes the fluid delivered by the inlet passageway 25 over thewhole upstream surface of the filter-adsorber assembly 12. To preventaggregates and other large obstructions from blocking flow at or nearthe junction of the inlet passageway 25 and the inlet plenum 22 and, atthe same time, to minimize hold-up volume in the housing 11, the depthof the inlet plenum 22 is greatest at the bottom of the housing 11 anddecreases along the vertical axis A to a minimum value at the horizontalcenterline of the housing 11.

The outlet section 16 of the housing 11 includes a circular outlet plate31 and a cylindrical collar 32 which extends from the periphery of thecircular outlet plate 31 to the periphery of the circular inlet plate20. The cylindrical collar 32 is preferably integrally formed with thecircular outlet plate 31 and joined to the circular inlet plate 20 inany suitable manner, e.g., by an adhesive or by sonic welding.

The inside surface of the circular outlet plate 31 defines a wall 33which faces the downstream surface of the filter-adsorber assembly 12.The wall 33 includes a plurality of generally concentric circular ridges34 which define concentric circular grooves 35. The ridges 34 abut thedownstream surface of the filter-adsorber assembly 12. The circulargrooves 35 collectively define an outlet plenum 36 which collects thefluid passing through the filter-adsorber assembly 12. The depth of theoutlet plenum 36 is made as small as possible to minimize hold-up volumewithin the housing 11 without unduly restricting fluid flow.

In accordance with another aspect of the invention, the wall 33 furtherincludes a passageway such as a slot 40 which communicates with theoutlet 14 at or near the top of the outlet section 16. The slot 40,which collects fluid from each of the circular grooves 35 and channelsthe fluid to the outlet 14, preferably extends from the bottom to thetop of the outlet section 16 along the vertical axis A. In the exemplaryseparation device 10, the width of the slot 40 remains constant but thedepth of the slot 40, which is greater than the depth of the outletplenum 36, increases from the bottom to the top of the outlet section 16along the vertical axis A. Alternatively, the height may be less thanthe diameter of the housing, the width may vary, or the depth may remainconstant. For example, the slot may extend from the top of the housingalong the vertical axis A a distance in the range from about 80% of theinside diameter of the housing.

The outlet 14 may be variously configured. However, the outlet 14 of theexemplary depletion device 10 includes a longitudinal outlet ridge 41which extends along the outside surface of the outlet plate 31 parallelto the vertical axis A. The lower end of the outlet ridge 41 may befashioned as a tubing connector or as a socket for receiving a tubingconnector or other apparatus. The outlet 14 further includes an outletpassageway 42 which communicates with the slot 40 at or near the top ofthe housing 11, extends through the outlet ridge 41, and opens at thelower end of the outlet ridge 41.

As blood starts to flow through the apparatus, filling it from thebottom and emptying at the top, air is displaced and flows towards andout of outlet passageway 42. By careful design of the exemplaryapparatus it has been possible to reduce, but not to eliminatecompletely, the situation in which some liquid reaches the area adjacentto the outlet passageway 42 before all of the air is cleared from theinner parts of the housing assembly. In the absence of slot 40, thislagging air flow would carry some red cell-containing suspension intothe outlet passageway 42. Slot 40 allows the blood so carried to flowinto the slot, where the air is harmlessly separated from the liquidsuspension. The air then rises harmlessly to the outlet 14 ahead of therising fluid level in the slot 40 and is almost completely ejectedbefore the liquid level reaches the top of the outlet plenum 36 andoutlet passageway 42. Thus, air is very efficiently cleared from thehousing 11 of the exemplary depletion device 10 according to theinvention. For example, in a depletion device which has an insidediameter of about 8.9 cm, an initial air volume of 36 cc, and a slotabout 8 cm high, about 0.73 cm wide, about 0.2 cm deep at the bottom,and 0.33 cm deep at the top, the residual volume of air passing throughthe outlet after 1 or 2 cc of blood has passed through the outlet isestimated to be less than about 0.1 cc.

In order to understand the importance of the slot and the flow passageconfiguration, the equivalent operation of a conventional leucocytedepletion unit will be described.

In conventional units, fluid enters at the top of the housing and exitsat the bottom. The housing of such a unit is typically connected byplastic tubing between a blood bag upstream from the conventionalhousing and a transparent drip chamber downstream from the conventionalhousing and thence to the patient. During priming, the housing alongwith the drip chamber is inverted and blood is forced through theconventional housing into the drip chamber. This has the disadvantagethat some pressure head is lost, but, more seriously, fluid reaches theexit of the conventional housing and enters the drip chamber while asmuch as 1 to 2 cc or more of air is still trapped in the conventionalhousing. Blood bank practice requires that the volume of air deliveredto the collection bag be kept to the lowest possible value, even 1 or 2cc being undesirable.

The filter-adsorber assembly 12 preferably comprises a number ofindividually preformed layers as described below under the heading"Fabrication of Fibrous Elements." During the development stage,housings were constructed for testing which incorporated the basicinternal configuration described above, but in addition were variablewith respect to the thickness of the filter-adsorber assembly. In thisway, it was possible to test filter-adsorber assemblies varying in totalthickness. In each case, the distance between the tips of the ridges 26,34 of the inlet and outlet sections was adjusted to be equal to thenominal total thickness of the filter-adsorber assembly.

To provide an interference fit of the filter-adsorber assembly 12 withinthe housing 11, the filter-adsorber elements were cut from largeprecompressed slabs to a diameter about 0.1 to about 0.5% larger thanthe inside diameter of the cylindrical collar 32. The filter-adsorberelements were cut in such a manner as to maintain true right cylindricalform at their outer edges. This, coupled with the slight oversizing,provides good edge sealing, i.e., an interference fit, between the outeredges of the filter-adsorber assembly 12, made up of the variousfilter-adsorber elements, and the inner periphery of the housing 11.

Fabrication of a Gel Prefilter Element

A first element of those assembled into the above described housing isreferred to as a gel prefilter. A proportion of PRC specimens containgels, fat globules, or microaggregates which can clog filter media. Thegels form a phase distinct from, and not miscible with, the blood plasmain which they are suspended. The state-of-the-art procedure for copingwith clogging of filters is enlargement of the pore openings of theupstream layer or layers, continuously or in relatively small steps, butthis procedure is inefficient when applied to the device of thisinvention, as a significant number of graduated pore size layers arerequired, and these tend to occupy a relatively large volume, and forthis reason would cause an excessive volume of blood to be held upwithin the device. Whereas the normal means calls for uniformlygraduated pore size, continuously or in relatively small steps, the porediameter of the preferred products of this invention change abruptly, bya factor of about ten, in the transition from the gel prefilter (firstelement) to the immediately adjacent microaggregate filter (secondelement), thereby accomplishing a substantial reduction in the overallvolume of the filter element.

Needle punched webs are made using staple fibers, which for syntheticfibers are usually derived from continuous filament by cutting ortearing the filament into lengths of usually about 3 to 6 cm. Thesestraight lengths are laid onto a moving belt after suspending them inair, and the fibers are interlaced by reciprocating multi-barbedneedles.

The fibers assume the form of irregular loops, circles, and spirals,interspersed with a variety of other irregular shapes. Straight sectionsare few, and fewer sections still are straight for more than a fractionof a millimeter. A notable characteristic is that at least about 90% ofthe fibers depart for at least one portion of their length from theplanar structure which characterizes other non-wovens, i.e., significantportions of the fibers of needle punched media are not parallel to theplane of the web. Gels appear to penetrate easily into this type of web,but to be effectively retained within the web, as may be seen bypost-test microscopic examination.

The structure of needle punched webs is in strong contrast with respectto fiber orientation when compared with non-wovens such as melt blownweb, in which the fibers are essentially parallel to the plane of theweb.

Needle punched webs are generally thicker as made than is desired forgel removal, and for optimal use are hot compressed to a controlledsmaller thickness. Fabric so made was discovered to be particularlyeffective in retaining gels. Further, such fabrics can be nearly filledwith collected gel, yet allow free flow of blood to the downstreamcomponent of the system.

While the gel prefilter does not recover microaggregates directly byfiltration, the gels it retains may contain microaggregates, and theseare efficiently retained along with the gels.

The "type A" gel prefilters used in the examples of this inventioncomprise a needle punched web made using polyester PET fibers of averagediameter about 23 μm, bonded by polyethylene isoterephthalate. Nominalweight is about 0.008 g/cm². The fiber lubricant is removed using a hotsolution of trisodium phosphate and detergent, and the web thenthoroughly washed and dried prior to use.

A needle punched web identical with the web described above was used asone of the components of the gel prefilter of U.S. Pat. No. 4,925,572.For use with PRC derived from freshly drawn blood which has relativelyfewer gels and microaggregates, the same prefilter has been used, butcompressed to a smaller thickness, as described below.

Other gel prefilters that can be used in the devices of this inventioninclude melt blown fibrous webs, particularly those having fiberdiameters of from about 10 to about 30 μm, and preferably those withfiber diameters of about 20 μm.

Fabrication of Preferred Microaggregate Filter

In U.S. Pat. No. 4,925,572, three layers of prefiltration are described.For use with fresh PRC, fewer prefiltration layers can be used, orindeed none at all need be used, with little or no risk of clogging.Among the Examples in accordance with this invention, we have used asthe microaggregate filter a 6.5 mg/cm² layer of web of fiber diameter3.2 μm followed by a 6.9 mg/cm² layer of web of fiber diameter 2.9 μm indiameter. These are compressed to a voids volume of about 74% to about84%. The fibers of both these layers are surface grafted to provide aCWST in the range of about 60 to about 70 dynes/cm.

Fabrication of an Adsorption/Filtration Element

Leukocytes are removed to only a small degree by the gel prefilter andmicroaggregate filter. The principal contributor to leukocyte removal isthe adsorption/filtration element, which comprises preferably one ormore hot compressed preforms of multiple identical layers of relativelysmall fiber diameter melt blown web.

Preforming and Assembly of the Elements

In a preferred filter of the invention, flow through the above elementsis in the order in which they are listed, that is, gel prefilter,microaggregate filter, and then the adsorption/filtration element. Thegel prefilter preferably comprises about two to four layers, themicroaggregate prefilter comprises preferably one to four layers, andthe adsorption/filtration element generally comprises one or morepreforms, each comprising a number of layers. In a more preferredembodiment, the adsorption/filtration element comprises two sets ofmulti-layers, each comprising a different voids volume. Multilayers maybe preferred for the adsorption/filtration element because the meltblowing process is such that making a single layer of the weight,thickness, fiber diameter and uniformity required is difficult.

These multiple layers can be used as individual preformed layersassembled in the order noted, however, it is sometimes more convenientto fabricate them as subassemblies. In one preferred configuration ofthe gel prefilter, two layers of needle punched medium and one of meltblown medium are hot compressed together into a single preform, while inanother two or more precompressed layers of melt blown web are used asseparate layers.

The values cited above and in the examples can be varied within limitswhile meeting the objective of this invention. To determine whether anyparticular variation produces a fully equivalent product, tests arerequired. Thus, it should be understood that, while the precisematerials, fiber diameters, weights, densities, thicknesses and numberof layers can be varied somewhat while achieving equivalent or possiblyeven better results, that which is disclosed herein is intended as aguide to the design of a device meeting the stated objectives of thisinvention and that devices made with such variations fall within thescope of this invention.

With the exception of the gel prefilter, all of the elements arepreferably surface treated to a CWST in excess of about 55 dynes/cm, butnot in excess of about 75 to about 80 dynes/cm, and more preferably fromabout 60 to about 70 dynes/cm.

Hot compressed element preforms made using melt blown fibrous mats whichhave been surface modified to raise their CWST values by 5 or moredynes/cm are palpably better with respect to firmness and resistance tofraying when compared with discs made by hot compression followed byradiation grafting. Grafting prior to hot compression is for this reasonpreferred; however, serviceable elements could be made by hotcompression followed by grafting.

While the examples of this invention have used hot compression to formthe integral elements which together combine to provide prefiltration,gel removal, and adsorption, it would be feasible to form the integralelements by other means, such as resin bonding, and a device utilizingthis or similar alternatives is within the scope of this invention.

Melt blown fibers have been preferred for use in all but the first layerof these devices. Should finer melt blown or other fine fibers, forexample, fibers made by mechanical fibrillation of larger diameterfibers or by other means, become available in the future, their use inelements for leucocyte depletion devices would be within the scope ofthis invention.

Edge Sealing the Preformed Elements into the Housing

The housing is preferred to be of generally disc form, or morerigorously stated, in part have the form of a right cylinder. Thepreformed elements are made also in right cylindrical form, of dimensionabout 0.1 to about 0.5% larger in diameter than that of the innersurface of the housing. When assembled, a good seal is obtained, with nodetectable bypassing during service.

In order to achieve good sealing, circular elements must have a trulyright cylindrical form. That form is not achieved by all the means bywhich the elements can be cut to circular form; for example, an obviousmeans, stamping out a circle using a steel rule die, does not provide anacceptable outer seal. Instead, the disc must be cut to its finisheddiameter using means which achieve the geometry of a true right cylinderat the cut edges. This has been achieved in the practice of thisinvention by construction of a circular knife of the required diameter,which is rotated to cut a true right cylinder while holding gently butfirmly in place the inner and outer surfaces of the precompressed slabfrom which the disc is cut.

Circular housings may be adapted to a procedure in which a bagcontaining the collected PRC is attached to the filter aseptically,followed by application of pressure to the bag containing the PRC toforce the PRC through the filter into a second collection bag.

In an alternate procedure the filter and a second PRC collection bag areprovided as part of the blood collection set prior to attaching the setto the blood donor. When this procedure is used, the blood is drawn intothe first collection bag, that bag along with the filter and theauxiliary bags are placed into the centrifuge bucket, following whichthe assembly is spun to make the PRC. For use in this procedure it isdesirable for reasons of economy to use as small as possible a bucket.In order to make this possible, it may be preferred that the filter havea form other than circular, for example, rectangular. Rectangularfilters can be sealed by an interference fit at their outer edge into arectangular housing, however, they may in addition or alternately bepreferred to have a peripheral compression seal.

CWST of the Elements

The gel prefilter (first) element may have a low CWST without harm, andindeed may function better in that condition. The results of tests inwhich sufficient PRC is run through a device to cause clogging or nearclogging, followed by dissection, inspection, and testing of thepressure drops of the individual layers, indicate that little if anyimprovement can be accomplished by increasing the CWST of this layer.The microaggregate filter element and the adsorption/filtration elementare preferably modified to a CWST of between about 55 to about 80dynes/cm, and more preferably to between about 60 and about 70 dynes/cm,and still more preferably to between about 62 and about 68 dynes/cm.

Red Cell Recovery

No significant changes in hematocrit were detected when the hematocritvalues for the blood in the bag were compared with the effluent from thedevices in accordance with this invention.

Some of the incoming blood or PRC is lost due to hold-up within thedepletion device. That loss is reported as blood hold-up volume.

Characterization of Porous Media by Physical Characteristics

Formulae have been proposed to predict pore diameter. These formulaetypically use fiber diameter, for example as determined by BET testing;bulk (apparent) density; and fiber density. One such, for example,calculates the average distance between fibers. However, the averagedistance between fibers can not be a meaningful predictor of performanceas in any liquid flow path it is the largest pore or pores encounteredwhich control performances, and this is particularly true of deformable"particles" such as leucocytes. In a fibrous mat such as made by meltblowing, the fibers are laid down in a random manner, and the pore sizedistribution is quite wide. Other means for forming fibrous mats, e.g.,air laying, or formation on a Fourdrinier screen, also produce wide poresize distributions. In these circumstances, the average distance betweenfibers is clearly a poor predictor of performance. A variety of otherformulae have been proposed to allow calculation of pore diameters fromdata on fiber diameter, fiber density and bulk density, but applicantsare unaware of any formula that has proved useful for calculating apriori the effective pore diameter of filters for liquid service.

Measurement of fiber surface area, for example by gasadsorption--popularly referred to as "BET" measurement--is a usefultechnique, as the surface area is a direct indication of the extent offiber surface available to remove leucocytes by adsorption. In addition,the surface area of melt blown PBT webs can be used to calculate fiberdiameter. Using PBT, of density 1.38 g/cc as an example: ##EQU3## thisvolume is equal to the fiber cross sectional area multiplied by itslength, hence ##EQU4##

Surface area of the fiber is πdL=A_(f) (2) ##EQU5## where L=total lengthof fiber per gram, d=average fiber diameter in centimeters, and A_(f)=fiber surface area in cm² /g.

If the units of d are μm, the units of A_(f) become square meters/gram(M² /g), which will be used hereinafter. For fibers other than PBT,their density is substituted for that of PBT.

A second characteristic necessary to describe a porous medium adequatelyto permit it to be reproduced is its pore diameter (Dp). We have used amodified OSU-F2 test for this purpose; this test and its mode of use aredescribed in the following section.

Other characteristics which describe a porous medium include apparent(bulk) density (ρ) in grams/cubic centimeter (g/cc), the fiber density(also in g/cc), the thickness (t) of the elements of the medium,specified in centimeters (cm), the cross sectional area available forflow through the filtering element in square centimeters (62 cm² for allof the examples), and the CWST in dynes/cm. Specifying these parametersdefines a filter or filter-adsorber element of predictable behavior whenused for leucocyte depletion.

Processing Blood Using the Products of this Invention

In current blood banking practice, where platelet concentrate (PC) isdesired to be recovered, the sequence of operation is:

a. Perform venipuncture and draw about 400 to about 450 ml of blood intoa sterile collector bag in which an anti-coagulant has been preplaced.This collection bag is attached by tubing to two other bags via a teeconnection, these bags denoted respectively as the platelet bag and theplasma bag.

b. The collector bag is then placed in a centrifuge and spun for about 3minutes at conditions which develops about 2000 G (i.e., 2000 times theforce due to gravity) during which the red cells are concentrated,forming the PRC at the bottom of the bag.

c. The collector bag is then placed in a device which is denoted as an"expresser", but in one commonly used version is denoted as a "plasmaextractor". The expresser squeezes the bag, developing about 1 to about1.5 psi of internal pressure. The operator opens a valve at the top ofthe bag, allowing the supernatant fluid containing the plasma and mostof the platelets to be decanted into the platelet bag, and leaving inthe collection bag about 170 to about 250 ml of PRC. It is this PRCwhich is, in a separate step, processed by the filter of this inventionin order to reduce its leucocyte content.

d. The remaining steps in the blood processing as generally practicedare preparation of plasma, or of platelet concentrate and plasma. Theseare not described, as they are not pertinent to this invention.

In alternate types of procedure, for example those designated as Adsolor SAG-M, the procedure of steps 1, 2 and 3 is similar except that aharder (higher G) spin may be used, and after decanting the supernatantliquid in an expresser, the red cells are resuspended in a physiologicalsolution containing saline and an anti-coagulant, forming the PRC.

In the practice of this invention, the collected PRC prepared by theseor similar processes is as the next step passed through the filter ofthis invention. This may be done in a separate step in which a filterand second collecting bag are aseptically attached to the PRC bag, andthe PRC is forced, for example by a pressure cuff developing a pressureof about 0.4 Kg/cm², to pass through the filter into the secondcollection bag. Alternatively the filter and the second collection bagmay be attached to the lower end of the whole blood collection bag priorto collecting the blood from the donor; then, after the supernatantfluid in the blood collection bag has been removed by centrifuging anddecanting, and while the bag is still in the expresser, the PRC istransferred through the filter into the second collection bag using thepressure provided by the expresser.

EXAMPLES

All tests run used blood drawn from human volunteers and processed usingeither Adsol CPDA-1 anticoagulant within 6 hours in accordance with thestandards of the American Association of Blood Banks to provide one unitof PRC. Hematocrits of the PRC were recorded and were with fewexceptions in the range of about 70 to about 80%, while hematocrits ofAdsol processed blood were generally in the range of about 55 to 65%.Leucocyte counts of the PRC prior to processing were in the range ofabout 1500 to about 17,000 per microliter (μL).

Priming time is defined as the time required to fill the test housingwith fluid.

Bag (i.e., influent) leucocyte counts were determined using a Model ZMCoulter Counter. Leucocyte counts are reported as number per microliter(μL). The conventional centrifugal method was used to determinehematocrits.

Use of automatic counters for the leucocyte depleted filter effluentsprovides incorrect results because automatic counters are designed to beoperated in the range of normal leucocyte content of whole blood and ofnormal PRC. Thus, the normal operating range of automatic counters isabout 10³ to about 10⁷ times the levels reached in the examples herein;as a consequence, automatic counter data at these low levels is quiteuseless.

A method to assay the degree of depletion of leucocytes to the very lowlevels of this invention, i.e., reduction of leucocyte count by a factorbetween about 10⁵ to about 10⁷ (99.999 to 99.99999% efficiency) hasbecome available only recently. The method was developed, in acooperative project with Pall Corporation (Pall), by the laboratory ofthe American Red Cross (ARC). Pall supplied the necessary highefficiency filters, while the ARC developed the assay method. This assaymethod, subsequently routinely practiced in the Pall blood laboratory,is described below.

Zeta potential was determined using a conventional streaming potentialapparatus.

The elements used in the examples were right circular discs, about 88.9mm in diameter at assembly. The stacked layers of elements, with a totalthickness of t cm were assembled into a housing as described above, witha clearance of about t cm between the faces of the two plenums, i.e.,between the tips of the ridges 26 on the inlet plate 20 and the tips ofthe ridges 34 on the outlet plate 31, as shown in FIG. 1. After piercingthe blood bag, leucocyte content was determined in the manner describedin the preceding part of this section.

Losses of red cells due to adsorption were, unless noted, too small tobe detected. For the examples of this invention losses due to hold-upwithin the filter housing were about 30 cm³ of PRC.

Pore diameters of filter media were determined using the modified OSU F2method and are reported as the diameter of hard particle at which about99.9% of the incident particles were removed. The F2 test used in makingpore size measurements is a modified version of the F2 test developed inthe 1970's at Oklahoma State University (OSU). In the OSU test, asuspension of an artificial contaminant in an appropriate test fluid ispassed through the test filter while continuously sampling the fluidupstream and downstream of the filter under test. The samples areanalyzed by automatic particle counters for their contents of five ormore preselected particle diameters and the ratio of the upstream todownstream count is automatically recorded. This ratio is known in thefilter industry as the beta ratio.

The beta ratio for each of the five or more diameters tested is plottedas the ordinate against particle diameter as the abscissa, usually on agraph in which the ordinate is a logarithmic scale and the abscissa is alog 2 scale. A smooth curve is then drawn between the points. The betaratio for any diameter within the range tested can then be read fromthis curve. Efficiency at a particular particle diameter is calculatedfrom the beta ratio by the formula:

Efficiency, percent=100(1--1/beta)

As an example, if beta=1000, efficiency=99.9%.

The removal rating cited in the examples presented herein is theparticle diameters at which beta=1,000, hence, the efficiency at theremoval ratings cited is 99.9%.

In the modified F2 test, efficiencies in particles in the range of fromabout 1 to about 20-25 μm were determined using as a test contaminant anaqueous suspension of AC fine test dust, a natural silicious dustsupplied by the AC Spark Plug Company. A suspension of the dust in waterwas vigorously mixed for three weeks until the dispersion was stable.Then, prior to use for measuring the pore size characteristics, thesuspension was passed through one of the gel prefilters of thisinvention, thereby removing oversize particles which would otherwisecollect on the filter surface and cause flow to stop. Pore diametervalues below 1 μm were obtained by extrapolation per the followingtabulation:

    ______________________________________    Beta Value at 1 μm                    Pore Diameter (μm)    ______________________________________    1000-2000       1    1200-1800       0.9    1800-2500       0.8    2500-4000       0.7    ______________________________________

Test flow rate was 100 liters per minute per square foot of filter areaand each pore size measurement as reported is the average of four tests.

The needle punched web used in the examples was scrubbed in order toremove the fiber lubricant, and then dried.

Preform thickness was measured using a 7.7 cm diameter anvil and with anapplied pressure of 4.3 g/cm².

Unless otherwise noted all of the elements used in the examples wereright circular discs of diameter 88.9 mm at assembly. Properties ofsub-sections of composite discs are listed in the order in which theblood flow through them.

Gel prefilter type A consisted of three layers. The two upper layersconsisted of 8±1 mg/cm² of 23 μm average fiber diameter PET needlepunched web, while the third and last layer consisted of a 7.7 mg/cm² 20μm average fiber diameter PBT melt blown web. The first of the threelayers was hot calendered to about 0.89 cm thick by passing the webbetween a pair of moving belts in an oven in order to heat the web toabout 165° to about 170° C., following which it passed throughcalendering rolls. The second and third layers were hot calendered inassembly to about 0.10 cm thick. All of the above were then hotcalendered together to a thickness of about 0.13 cm. The resulting gelprefilter consisted of 3 integrated layers of approximate density,respectively, 0.14, 0.19, and 0.32 g/cm², the last layer having a porediameter of about 20 to about 30 μm.

Gel prefilter type B consisted of four adjacent layers of about 20 μmdiameter fiber melt blown web each separately hot calendered in themanner described above to obtain the characteristics listed below:

    ______________________________________    Weight        Thickness Voids    mg/cm.sup.2   cm        Volume, %    ______________________________________    3.1           .025      91    4.1           .025      88    5.7           .025      84    7.7           .025      78    ______________________________________

Although the thickness of the separately measured layers of the type Bprefilter add up to about 0.1 cm, the total thickness when the four werestacked on each other was about 0.08 cm. The last layer had a porediameter of about 20 to about 30 μm.

All type B gel prefilters were made using PBT fibers with no surfacemodification.

The development procedure went through stages at which first alaboratory (L) method was used to achieve hot compression, and later aproduction (P) method was used to achieve the same purpose. In the Lmethod the necessary microaggregate filter and adsorption filter layerswere assembled as a stack and the whole compressed between Teflon linedaluminum alloy plates at 165° C. for about 40 seconds. In the P method asimilar stack was passed between two moving Teflon coated belts heatedto about 165° to about 170° C. for a period of about 30 seconds,following which they were passed through a pair of calender rolls toachieve the desired density or thickness.

The filter effluents were assayed to determine leucocyte content using a"Ficoll" method, alternatively referenced hereinafter as the "ARC"method, in which the leucocytes are separated in concentrate formenabling a high proportion of all of the leucocytes present to becounted directly. This method was developed in the laboratory of theAmerican Red Cross. Prior to this development no assay procedure wasavailable which was capable of counting the very low levels ofleucocytes, less than about 10² to about 10⁵ per unit of PRC, which areobtained using the products of this invention. The Ficoll Assay isdescribed below:

1.0 Purpose

1.1 This test method is used to separate leucocytes from filtered packedred cells and determine the log efficiency of a leucocyte depletingfilter.

2.0 Materials and Equipment

one unit of fresh PRC or of whole blood

5 mL disposable polypropylene test tubes with caps

600 mL transfer bags

filter

pressure infusor

hemastat clamps

500 mL graduated cylinder

Ficoll solution (see section 4.4)

60 mL disposable polypropylene syringe

plasma extractor

Sorvall RC-3C General Purpose Refrigerated Centrifuge

blood bank pipets

vacuum suction apparatus

1% ammonium oxalate solution (see section 4.3)

aliquot mixer

Neubauer hemacytometer

plain hematocrit tubes

Fluorescent microscope with phase contrast and 20 and 40X objectives

250 mL disposable polypropylene centrifuge tubes

15 mL disposable polypropylene centrifuge tubes

hematology control: normal level and low abnormal level--Counter-Check™,Diagnostic Technology, Inc.

Acridine orange fluorescent stain (see Section 4.4)

3.0 Procedure

3.1 Mix the PRC unit and withdraw a sample in a 5 mL tube for use tocount influent leucocytes.

3.2 Connect the pre-weighed 600 mL transfer bag to the filter outlet,and the filter inlet to the blood bag.

3.3 Prime the filter with PRC using a pressure infusor set at 300 mm Hg.

3.4 When flow has begun, lower pressure to 200 mm Hg for remainder offiltration.

3.5 When filtration is complete, turn pressure off, clamp the collectionbag and remove it from the filter.

3.6 Determine the effluent volume by weighing the collection bag,subtracting the empty bag weight, and dividing by 1.08 (the density ofPRC). Record this volume.

3.7 Adjust the volume to 300 cc by discarding the excess, or by addingsaline, then remove the syringe and seal off the bag.

3.8 With a graduated cylinder, measure 300 cc of Ficoll solution and addit to the collection bag using a 60 mL syringe attached to the transferbag tubing.

3.9 Vigorously mix the Ficoll-blood solution and place the bag in aplasma extractor.

3.10 Clamp the tubing and remove the syringe.

3.11 Apply the extractor clamp and let the blood settle for 30 minutes.

3.12 Carefully express the upper layer into a 250 mL centrifuge tube byopening the hemastat clamp. Do not disturb the interface whileexpressing the maximum amount of Ficoll.

3.13 Release the extractor clamp and repeat step 3.8 using a sufficientvolume of Ficoll to fill the 600 ml bag, then repeat 3.9, 3.10, 3.11,and 3.12.

3.14 Centrifuge the tubes at 775 g for 15 minutes at room temperature inthe Sorvall™ centrifuge.

3.15 When spin is complete, use a blood bank pipet attached to a vacuumflask to extract and discard the supernatant leaving a red pellet.

3.16 Resuspend the pellet in 250 mL of 1% ammonium oxalate.

3.17 Allow the suspension to mix on an aliquot mixer for 10 minutes tolyse the red blood cells.

3.18 Centrifuge the tube at 432 g for 10 minutes at room temperature anddiscard the supernatant as before.

3.19 Resuspend the pellet in 2-3 mL of ammonium oxalate using a bloodbank pipet to draw the pellet up for mixing. Transfer this suspension toa 15 mL polypropylene centrifuge tube combining all pellets from oneblood unit.

3.20 Fill the tube to the 15 mL mark with the 1% ammonium oxalatesolution, mix, and allow tube to set for 10 minutes.

3.21 Spin the 15 mL tube in the centrifuge at 775 g for 10 minutes atroom temperature.

3.22 Decant the supernatant down to the 0.5 mL line on the 15 mL tube.Carefully resuspend the pellet using a pipetter. Add 0.05 mL of AcridineOrange stain to the suspension, and weigh and record the tube weight todetermine the final volume of the suspension.

3.23 Leucocyte Counts: All counts are performed manually

3.23.1 Control Counts

3.23.1.1 Control counts are performed daily by making a 1:100 dilutionof each control in 1% ammonium oxalate.

3.23.1.1.1 Add 0.01 mL of the control to 0.99 mL of 1% ammonium oxalateusing an adjustable pipetter. Mix well and let the dilution sit for atleast 10 minutes to lyse the red blood cells, then add 0.05 mL ofAcridine Orange.

3.23.1.1.2 Charge each side of a hemacytometer using a plain capillarytube, taking care not to overload or underload the chamber.

3.23.1.1.3 Allow the hemacytometer to sit in a moist atmosphere (coveredpetri dish with moistened filter paper in bottom half of the dish) forten minutes.

3.23.1.1.4 Count the number of leucocytes in the nine large squares onboth sides of the hemacytometer using the phase contrast UV microscope.

3.23.1.1.5 Record the counts from each side (each nine squares) of thehemacytometer.

3.23.1.1.6 To determine the number of leucocytes/μL, use the followingformula: ##EQU6##

3.23.2 Influent Leucocyte Counts

3.23.2.1 Influent leucocyte counts are performed using the sameprocedure as for the control counts except that a total of 36 largesquares (two hemacytometers) are counted.

3.23.2.2 To calculate the leucocytes/μL, use the same formula as above.If 36 large squares are counted, the total number of cells×28=cells/μL.

3.23.2.3 The number of leucocytes/μL×1000 equals the number ofleucocytes/mL.

3.23.2.4 Multiply the number of leucocytes/mL by the effluent volume todetermine the total leucocytes in the prefiltration sample (note: theeffluent volume is used in this calculation, not the influent volume,because log reduction is a direct volume/volume comparison and does nottake into account the hold-up volume).

3.23.3. Effluent Leucocyte/Counts

3.23.3.1 Effluent leucocyte counts are performed using he undilutedfinal ammonium oxalate suspension from step 3.24.

3.23.3.1.1 Charge the hemacytometer and count as before.

3.23.3.1.2 If the number of cells counted on both sides of thehemacytometer is 30 or less, continue counting hemacytometers until 30cells or 5 hemacytometers are counted.

3.23.3.2 Determine the total leucocytes in the post-filtration sample asfollows:

3.23.3.2.1 Divide the effluent volume by the volume of the finalsuspension to determine the concentration.

3.23.3.2.2 Use the following formula to calculate leucocytes/μL:##EQU7##

3.23.3.2.3 The number of leucocytes/μL×1000 equals the number ofleucocytes/mL.

3.23.3.2.4 Multiply the number of leucocytes/mL by the effluent volumeto get the number of leucocytes in the postfiltration sample.

3.23.3.2.5 The Ficoll procedure gives a nominal 53% yield of leucocytes,thus the number from the step above must be divided by 0.53 to determinethe total leucocytes in the postfiltration sample.

3.24 Determine the log reduction by dividing the total number ofleucocytes in the prefiltration sample by the total number of leucocytesin the postfiltration sample and taking the log of this quotient.

4.0 Supplementary Information

4.1 Ficoll Formula

    ______________________________________    100.0     g          Ficoll* 400 DL    20.0      g          Bovine Serum Albumin    2000.0    mL         Stock EBSS (see below)    Mix the above ingredients in a 2 L volumetric flask    and warm to 37° C. while stirring. Filter solution    through a 1.2 μm filter disc and then through a    0.45 μm filter disc. Store at 4° C.    ______________________________________     *Ficoll 400 DL is a dialyzed, hydrophilized, synthetic polymer of sucrose     with a molecular weight of approximate 400,000 available from Sigma     Chemical Co.

4.2 Stock EBSS Formula

200.0 mL Earle's Balanced Salt Solution (10×)

1800.0 mL deionized H₂ O

40.0 mL Hepes Buffer Solution

Mix the above ingredients and store at 4° C. Warm to room temperatureprior to use.

4.3 Ammonium Oxalate Formula

10.0 g Ammonium Oxalate

0.10 g Thimerosal

0.43 g KH2PO4

0.57 g Na2HPO4

Mix the above ingredients in a 1 L volumetric flask and dilute to 1 Lwith deionized water. Check the pH and adjust to 6.8 if necessary. Storeat 4° C. Warm to room temperature prior to use.

4.4 Acridine Orange Stain Formula

4.4.1 Stock Acridine Orange (1000×solution)

6.69 mg Acridine Orange/ml of DI water

Store in the dark at 2°-5° C.

4.4.2 Working Acridine Orange (10×solution)

dilute Stock Acridine Orange with Stock EBSS (see 4.2)

good for 1 month if stored in the dark at 2°-5° C.

The above described assay method has a nominal recovery efficiency of53% of the leucocytes actually present in the filtered PRC. Theconsistency of the 53% figure is about +15 to -25%; however, the logreduction calculated is affected in only a minor way by these deviationswhen a single test is run, and consistency is improved by running fouror more tests on each filter tested.

The leucocyte removal efficiencies obtained for the products of thisinvention using the ARC assay procedure can be stated in severalalternate ways. Using as an example a unit of PRC containing 10³leucocytes in the total filtrate, and a value of 10⁹ leucocytescontained in an equal volume of the PRC prior to filtration, then theratio of effluent content/influent content is 10³ /10⁹ =10⁻⁶, andefficiency of leucocyte removal can be reported as:

(a) 100(1-10⁻⁶)=99.9999% or

(b) leucocyte content is reduced by a factor of one million (1/10⁻⁶=10⁶) or

(c) log reduction=-6

A convenient method is to use the term log reduction and omit the minussign, since minus is inferred by the term "reduction". This nomenclatureis widely used, and we will use it when reporting efficiencieshereinafter.

Examples 1-6 were performed in the ARC laboratory using preformed filterelements prepared in the inventors' laboratory by Pall Corporationpersonnel using materials and procedures conceived and developed by theinventors with no input from ARC. ARC's contribution to this part of thedevelopment was confined to developing and initially performing theFicoll test on filters developed and made by Pall, and then reportingtest results to the Applicant. Later the Ficoll test was performed inPall's laboratory using Pall personnel.

This group of examples consisted of a single preform made using 2.6 μmfiber diameter melt blown PBT web, hot compressed using the L(laboratory) technique described above. No prefiltration was used. Thetests were run using 47.6 mm diameter discs, assembled into Pall madehousings of slightly smaller diameter. Each test was run using onequarter of a unit of fresh PRC of hematocrit 50 to 55%. Flow rate of 4to 8 cc per minute was obtained at a pressure of 40 inches of fluidhead. The resulting data for these examples is listed in Table 1. Whenplotted, 5 of the 6 points fall on or near to a straight linerepresented by the equation ##EQU8## where ρ is the density in grams/cc,and the weight of the adsorption/filtration element is

    (0.5ρ-0.029)g/cm.sup.2                                 (2)

This equation provides guidance for selecting the densities of PBTfilters which have a desired average log reduction between about 4.5 toabout 7; however, filters with log reduction in the upper part of therange, with voids volumes less than about 74% to about 78%, may becomeclogged prior to passage of a single unit of PRC, consequently gel,microaggregate filters, or multilayers may be required to preventoccational clogging.

In example 7, ten discs with a pore diameter of 0.9 μm duplicating thoseof Example 4, except 88.9 mm in diameter, are assembled into an 88.6 mmdiameter housing with ridge to ridge depth of 0.439 cm. Priming isaccomplished in 50 to 100 seconds at a pressure of 0.4 Kg/cm², and onefull unit of fresh PRC of hematocrit of 70 to 80% derived from bloodcollected into CPDA-1 anticoagulant is passed through each at a pressureof 0.27 Kg/cm². The average time to pass one unit through five of theten discs ranges from 15 to 25 minutes. The average log reduction isapproximately 6.4. Flow in the other five of the ten tests will fallwithin a 2 hour period to less than 0.7 cc/min, and these tests are thendiscontinued.

In Example 8, ten integral preformed discs duplicating those of Example7 are assembled together with a type A gel prefilter into an 88.6 mmdiameter housing with ridge to ridge depth of 0.569 cm, and one unit offresh PRC will be passed through each. The total volume of one unit ofPRC is passed at a pressure of 0.27 Kg/cm² in 15 to 25 minutes. Theaverage log reduction is approximately 6.4.

In Example 9, ten discs duplicating those of Example 7 are assembledtogether with a type B gel prefilter into an 88.6 mm diameter housingwith ridge to ridge depth of 0.519 cm, and one unit of fresh PRC ispassed through each in the same manner as in Example 7. The total volumeof PRC is passed in 15 to 25 minutes. The average log reduction isapproximately 6.4.

In Examples 8 and 9 the addition of the type A and B prefilters willmake clogging less likely than would be the case if they were not used,as in Example 7.

Example 10 is prepared in the same way as Example 9, except that theadsorption/filtration element is integral with a microaggregate filter,formed by including two layers of media of larger fiber diameter in theupstream side of the lay-up, followed by hot compression to obtain apreformed integral element. Specifically, the uppermost layer is madeusing 3.2 μm diameter fibers in the form of a mat of weight 0.0065g/cm², and the adjacent layer is made using 2.9 μm diameter fiber ofweight 0.0069 g/cm², while the balance, the adsorption/filtrationelement consists of multiple layers, all of fiber diameter 2.6 μm. Totalweight is 0.130 g/cm² compressed to a voids volume of 76.5%(density=0.324 g/cc) and thickness of 0.439 cm. The filter so made willpossess capability for microaggregate removal, and be more resistant toclogging, as would otherwise be caused by the occasional specimen ofdonated blood which contains an unusually high content ofmicroaggregates. The properties of the filter of Example 10 with respectto efficiency of leucocyte depletion and blood transit time are notsignificantly altered from those of Example 9, as the fiber surface areaof Example 10 is reduced from that of Example 9 by only about 1%. Thepore sizes obtained by F2 testing are essentially equal for the twoexamples. Blood hold-up volume within the voids of the gel prefilter,the microaggregate prefilter, and the adsorption/filtration element are,respectively, 3.9 cc, 1.6 cc, and 19.1 cc for a total of 24.6 cc.

Examples 11 to 14, summarized in Table 2, illustrate the use ofequations (3) and (4) set forth above to calculate the manner in whichfilter assemblies may be made which have lower efficiencies, but whichduring filtration pass PRC at a lower pressure drop, making it possibleto use gravity to induce flow at a satisfactory rate when processingblood with relatively high hematocrit. All contain integral elementscombining microaggregate elements with adsorption/filtration elements,with the fibers surface modified to a CWST of 60 to 70 dynes/cm, andpreferably 62 to 68 dynes/cm. The device of Example 11 also incorporatesa type B gel prefilter.

Other variations of density and thickness are possible. All of theExamples 11-14 can be made with a higher density (i.e. lower voidsvolume), while retaining equal or better leucocyte removal efficiency.Thus, Example 15 is made in the same manner as Example 11 except thatthe voids volume is changed to 76.5%. The resulting log leucocytereduction will be intermediate between 6 and 6.5, and the blood hold-upvolume within the element is reduced by about 10%. Similarly, Example 16is the filter of Example 12 with the voids volume decreased from 80.4 to78.5%, with log reduction between 5.5 and 6, and blood hold-up volumereduced by about 10%, and Example 17 is the filter of Example 13 withvoids volume decreased from 82.4 to 80.4%, with log reduction between 5and 5.5, and with blood hold-up volume decreased by about 10%. Example18 is the filter of Example 14 with voids volume decreased from 84.3 to82.4%, with log reduction between 4.5 and 5, and with blood hold-upvolume decreased by about 11%.

The void volume of each of Examples 16, 17, and 18 could be furtherdecreased, obtaining in this way equal or higher efficiency along withstill lower blood hold-up volume compared with Examples 16, 17, and 18.

Example 19 is essentially the filter of Example 10 with voids volumedecreased further. A type B prefilter was used in this construction inorder to minimize the chances of clogging. Immediately downstream of theprefilter was a section consisting of nine layers, all of fiber diameter2.6 μm, with a CWST of 66 dynes/cm and a total weight of 0.054 g/cm²,compressed to a voids volume of 77% (density=0.321 g/cc) and a thicknessof 0.168 cm. Another section, downstream of this one, consisted of 20layers of the same type of fibers, however, compressed to a lower voidsvolume. The total weight of this section was 0.118 g/cm², voids volumewas 70% (density=0.414 g/cc), and thickness was 0.285 cm. When testedwith blood in accordance with the previous examples, this combinationgave a leukocyte reduction of 6 log, and a total filtration time of 32minutes.

It was previously thought that the use of voids volumes much below about74% would pass PRC more slowly and thus would tend to clog with higherfrequency. However, another surprising feature of this invention is thatvoids volumes of 70% were found to be quite suitable and efficient, andvoids volumes as low as 60% may be useful for some special applications.When lower voids volumes are utilized, it is found that it is thecombination of voids volumes with other factors, i.e., the number ofmultilayers in each section, rather than a particular voids volume byitself, that produces an element that is suitable in carrying out theinstant invention.

While the examples all deal with PRC, generally equivalent results willbe obtained when anticoagulated whole blood is used.

An extraordinary and very surprising feature of this invention is theability of red blood cells to pass through a filter of pore diameterless than 1 μm without apparent injury and no apparent losses. Theability to use such small pored filters was not anticipated, and wasseen only as a very unlikely possibility meriting exploration in theabsence of other approaches to the preferred goal of 100,000 to1,000,000 fold reduction of leucocyte content in a filter so small thatred cell loss due to hold-up is less than 10% of the average volume of aunit of PRC.

Red cell loss can be reduced by passing saline post filtration into orthrough the filter. Passing a volume about equal to that of the filterhelps to reclaim blood. It is generally a less desirable procedure toflush the filter with larger volumes of saline, as this undesirablyincreases blood volume and may reduce efficiency by flushing out somewhite cells. Use of saline requires manipulation which is relativelycostly in terms of labor, while at the same time compromising sterilityof the red cell concentrate. For these reasons, small hold-up volume,with correspondingly small red cell loss, is very desirable, as itobviates the need for using saline.

The adsorption/filtration elements described in the examples of thisinvention all comprise a non-woven web of average fiber diameter 2.6 μm.Of the various media available to the inventors, this was selectedbecause it can be made in large quantity with very reproducibleproperties. Should smaller fiber diameter webs become available in thefuture, or if such exist elsewhere at this time, these may be adapted byone versed in the art of filter development to be used in the mannerdescribed above possibly with advantage. A product so developed wouldfall within the scope of this invention.

Products resembling and similar in function to those of this inventionmay be made using coarser fibers. Such a product may perform similarlyin a general way to the product of this invention, and would fall withinthe scope of this invention.

Similarly, other materials and methods of surface modification may beused to achieve similar results, but these also would be within thescope of this invention.

                                      TABLE 1    __________________________________________________________________________    Adsorption/Filtration Element Only (No Prefiltration) -    2.6 μm dia. fiber                Blood                          Pore                                                   Average         No.            CWST                hold-up                     Zeta        Thick-   Voids                                               dia.,                                                   Leukocyte    Example         of dynes/                volume,                     Potential                            Weight,                                 ness,                                     Density                                          Volume,                                               micro-                                                   log    No.  Tests            cm  cc** millivolts                            g/cm.sup.2                                 cm  g/cc %    meters                                                   Reduction    __________________________________________________________________________    1    4  63  20.0 -30 to -40                            .103 .399                                     .259 81.2 1.2 5.4    2    8  63  19.2 -30 to -40                            .092 .379                                     .244 82.3 1.4 4.4    3    8  63  19.7 -30 to -40                            .089 .384                                     .233 83.1 1.5 4.8    4     8*            70  20.7  -2 to -12                            .143 .439                                     .324 76.5 0.9 6.4    5    4  70  21.5  -2 to -12                            .123 .439                                     .281 79.6 1.2 6.0    6    7  70  19.1  -2 to -12                            .104 .386                                     .269 80.5 1.2 5.5    __________________________________________________________________________     *Clogging prior to complete passage of the PRC was observed in 4 of eight     tests.     **Internal voids volume of the disc, calculated for a full size 88.6 mm     diameter disc.

                                      TABLE 2    __________________________________________________________________________    Filters with log reduction 4.5 to 6, and with reduced internal blood    hold-up                                   7    2         3     4    5    6    Blood    Properties of the filter/adsorber-                                   hold-up    microaggregate preform         volume    1                    Voids     within 8    Example         Weight,              Thickness,                    Density,                         Volume,                              Pore dia.,                                   filter Log    No.  g/cm.sup.2              cm    g/cc %    μm                                   elements, cc                                          Reduction    __________________________________________________________________________    11   .119 .400  .297 78.5 1.0   24.4* 6    12   .106 .392  .270 80.4 1.2  19.7   5.5    13   .092 .379  .243 82.4 1.4  19.4   5.0    14   .079 .366  .216 84.3 2.0  19.1   4.5    __________________________________________________________________________     *Includes gel prefilter type B

We claim:
 1. A device for the depletion of the leukocyte content of ablood product comprising a fibrous leukocyte adsorption/filtrationfilter having a pore diameter of from about 0.5 to about 2 μm, anegative zeta potential, and a CWST of from about 60 to about 70dynes/cm.
 2. A device according to claim 1 which further comprises amicroaggregate filter upstream from the fibrous leukocyteadsorption/filtration filter.
 3. A device according to claim 1 in whichthe fibrous leukocyte adsorption/filtration filter is disposed in ahousing having an inlet and an outlet and defining a blood product flowpath between the inlet and the outlet, wherein said filter is disposedwithin the housing across the blood product flow path, and wherein thedevice has a total voids volume of less than about 30 cc.
 4. A deviceaccording to claim 1 in which the fibers of the fibrous leukocyteadsorption/filtration filter have been radiation grafted with a monomercontaining a hydrophilic group.
 5. The device of claim 1 wherein thefibrous leukocyte absorption/filtration filter is preformed tocontrolled density and pore diameter.
 6. The device of claim 1 whereinthe filter has a CWST of from about 62 to about 68 dynes/cm.
 7. Thedevice of claim 1 wherein the filter further comprises at least one of agel prefilter and a microaggregate filter.
 8. The device of claim 1wherein the filter has a voids volume in the range of about 65% to about84%.
 9. The device of claim 1 wherein the filter has a pore diameter ofabout 2 μm.
 10. The device of claim 1 further comprising a fibrous gelprefilter and a fibrous microaggregate filter.
 11. The device of claim10 wherein the fibrous microaggregate filter is interposed between thefibrous gel prefilter and the fibrous leukocyte adsorption/filtrationfilter.
 12. The device of claim 1 wherein the fibrous leukocyteadsorption/filtration filter includes fibers having an average diameterof about 3 μm or less.
 13. The device of claim 1 wherein the fibrousleukocyte adsorption/filtration filter has fibers that have beenradiation grafted with at least one acrylic monomer;
 14. The device ofclaim 13 wherein the fibrous leukocyte adsorption/filtration filter hasfibers that have been radiation grafted with first and second acrylicmonomers.
 15. The device of claim 14 wherein the first acrylic monomeris methyl acrylate or methyl methacrylate.
 16. A device for thedepletion of the leukocyte content of a blood product comprising:afibrous microaggregate filter; and a leukocyte adsorption/filtrationfilter having a pore diameter of from about 0.5 to about 2 μm, anegative zeta potential, and a CWST of from about 55 to about 80dynes/cm.
 17. The device of claim 16 wherein the leukocyteabsorption/filtration filter is compressed to an average voids volume ofabout 60% to about 85%.
 18. The device of claim 17 wherein the leukocyteabsorption/filtration filter is compressed to an average voids volume ofabout 65% to about 74%.
 19. A device according to claim 16 in which theleukocyte adsorption/filtration filter is hot compressed to an averagevoids volume of about 65% to 84%.
 20. A device according to claim 16 inwhich the fibrous microaggregate filter comprises a fibrous webcompressed to an average voids volume of about 74% to about 84%.
 21. Thedevice of claim 16 wherein the leukocyte adsorption/filtration filterhas a pore diameter of about 2 μm.
 22. The device of claim 16 whereinthe fibrous microaggregate filter and the leukocyteadsorption/filtration filter each include multilayers.
 23. The device ofclaim 22 wherein the leukocyte adsorption/filtration filter includesnine or more layers.
 24. The device of claim 22 wherein the leukocyteadsorption/filtration filter includes two sets of multilayers.
 25. Thedevice of claim 22 wherein the fibrous microaggregate filter has a CWSTof from about 55 to about 80 dynes/cm.
 26. The device of claim 25wherein the leukocyte adsorption/filtration filter has a CWST of fromabout 60 to about 70 dynes/cm.
 27. The device of claim 26 wherein theleukocyte adsorption/filtration filter has a CWST of from about 62 toabout 68 dynes/cm.
 28. The device of claim 16 wherein the leukocyteadsorption/filtration filter is capable of depleting the leukocytecontent of whole blood.
 29. The device of claim 16 wherein the leukocyteadsorption/filtration filter is capable of depleting the leukocytecontent of fresh whole blood.
 30. The device of claim 16 furthercomprising a housing including an inlet and an outlet and defining ablood product flow path between the inlet and the outlet, said fibrousmicroaggregate filter and said leukocyte absorption/filtration filterdisposed within the housing across the blood product flow path, whereinthe fibrous microaggregate filter and the leukocyteabsorption/filtration filter are arranged to deplete the leukocytecontent of the blood product by a factor of at least about 30,000. 31.The device of claim 16 wherein the leukocyte adsorption/filtrationfilter has fibers that have been radiation grafted with a monomercontaining a hydrophilic group.